Gastrointestinal (GI) motility/functional disorders affect up to 25% of the US population. Common intestinal motility disorders include Irritable Bowel Syndrome and Fecal Incontinence, whereas more rare forms such as Hirschsprung's Disease have a genetic basis and are associated with absence or paucity of enteric nerves 1,2. Current treatment plans for GI motility/functional disorders range from changes in diet to bowel resection, however there are very few drugs available that target the primary deficiencies in controlled peristalsis. One barrier to research of GI disease is that it has largely relied on in ivo animal studies, which are intrinsically low throughput. Recently, we have established culture systems to generate human gastrointestinal tissue organoids through directed differentiation of human embryonic and induced pluripotent stem cells (collectively called PSCs). HIOs are three-dimensional structures containing most epithelial and mesenchymal cell types found in the intestine, but lack an enteric nervous system (ENS). However we recently demonstrated that the enteric nervous system could be built into intestinal organoids by introducing neural crest stem cells (NCSC) into the differentiation process (Workman et al., in preparation). We have used multiple genetic markers and genetically modified pluripotent stem cell (PSC) lines to demonstrate that ENS neurons have functional capacity in vitro, that HIOs contain multiple neuronal and glial cell types, and that neuroglial cells that self organize, along with smooth muscle cells, around the mucosa. Engraftment of HIOs + ENS into mice led to extensive maturation including formation of two neural plexuses that integrate into submucosal and myenteric muscle layers. Moreover, electrical stimulation of in vivo grown organoids resulted in peristaltic-like contractions that could be blocked with the neuronal blocker tetrodotoxin. In thi application we propose to incorporate an ENS into other regional organoids, including recently established gastric and colonic organoids, and to generate live-imaging reporter systems to study ENS function in vitro and in vivo.